The Study On Construction And Immunogenicity Of Swine Transmissible Gastroenteritis Virus DNA Vaccine

Porcine transmissible gastroenteritis(TGE) is a highly contagious,enteric disease of swine caused by transmissible gastroenteritis virus(TGEV).The disease is especially severe in baby pigs less than two weeks old,characterized by vomiting,severe diarrhea, and nearly 100%mortality.In present study,TGEV DNA vaccines were explored using TGEV S gene,N gene as target genes and porcine granulocyte-macrophage colony stimulating factor gene(pGM-CSF),porcine interleukin-6(pIL-6) gene as genetic adjuvants.The main contents were as follows:1.Cloning of TGEV S gene,N gene and the eukaryotic expression plasmids constructionThe 5' fragment of the TGVE SC-H strain S gene that encompasses all the four major antigenic domains and complete N gene were amplified by RT-PCR and cloned into pMD-19T vector;the recombinants were designated as 19T-S and 19T-N.Nucleotide sequencing and analysis showed that the amplified S gene and N gene were 2127 bp and 1149 bp in length,which respectively encode 708 and 382 amino acids.The results of sequence alignment showed that the amino acids sequence deduced from TGEV SC-H strain S gene and N gene respectively shared 95.5%-99.7%and 98.2%-100%in homology with other TGEV strains.The eukaryotic expression plasmids pVAX-S,pVAX-N and pVAX-S-N,which expressing or fusion expressing TGEV S and N gene,were constructed using pVAX1 as vector,pVAX-S,pVAX-N and pVAX-S-N were transiently tranfected into COS-7 cells and the expression of recombinant plasmids were confirmed by indirect immunofluorscence assay.The results showed that the eukaryotic expression plasmids were constructed correctly and the transfected COS-7 cells displayed specific immunofluorscence.The successful construction of the pVAX-S,pVAX-N and pVAX-S-N provide foundation for further research of TGEV DNA vaccines.2.Cloning of porcine granulocyte-macrophage colony stimulating factor and porcine interleukin-6 gene and study on their expressionThe full-length porcine granulocyte-macrophage colony stimulating factor(pGM-CSF) and porcine interleukin-6(pIL-6) gene were cloned from ConA stimulated porcine peripheral blood lymphocytes using RT-PCR and inserted into pMD19-T vector to construct 19T-GM and 19T-IL6.Nucleotide sequencing and analysis showed that the ORF of cloned pGM-CSF and pIL-6 gene were 435 bp and 639 bp in length,which respectively encode 144 and 212 amino acids.The recombined prokaryotic expression plasmids,which encoding of mature pGM-CSF and pIL-6 were transformed into Escherichia coli BL21 (DE3).High level expression of rpGM-CSF and rpIL-6 recombinant proteins were obtained and polyclonal antibodies against the recombinant proteins were prepared.The pGM-CSF and pIL-6 genes were released from the recombinant plasmids 19T-GM and 19T-IL6 and inserted into pVAX1 to construct eukaryotic expression plasmids pVAX-GM and pVAX-IL6.Plasmids were tranfected into COS-7 cells,the expression of pVAX-GM and pVAX-IL6 were confirmed by Western-blot.The successful construction of the pVAX-GM and pVAX-IL6 provide foundation for using them as genetic adjuvants of DNA vaccines.3.Study on immunogenicity of TGEV DNA vaccineSix-week old NIH mice were immunized intramuscularly three times at two weeks interval with TGEV DNA vaccine of pVAX-S,pVAX-S-N,pVAX-S+pVAX-N, pVAX-S+pVAX-IL6 and pVAX-S+pVAX-GM.The anti-TGEV IgG antibodies in murine serum were detected by indirect ELISA at 14,35 and 42 days post immunization.The results showed that specific anti-TGEV antibodies were induced in all test groups.The antibody level of pVAX-S+pVAX-N group was higher than pVAX-S and pVAX-S-N groups,the difference was extremely significant(P＜0.01) at 35 and 42 days post immunization.The antibody level of pVAX-S group was higher than pVAX-S-N group throughout the immunization and there existed significant(P＜0.01) different at day-42 post immunization.As genetic adjuvant,pVAX-IL6 and pVAX-GM showed promoting effect on antibody level stimulate by pVAX-S,pVAX-S+pVAX-IL6 group was higher than pVAX-S+pVAX-GM group,the difference was significant(P＜0.05) at day 35 and 42 post immunization.At 42 days post immunization,the percentage of CD3+,CD4+ and CD8+ T-lymphocytes from murine peripheral blood of different immunized group were detected by flow cytometry.The results showed that pVAX-S could effectively induce cellular immune responses in mice.Co-administration pVAX-N,pVAX-IL6 and pVAX-GM with pVAX-S strengthen the cellular immune responses with varying degrees, pVAX-N showed better cellular immune enhancement contribution than pVAX-IL6 and the later better than pVAX-GM.4.Oral immunization of attenuated Salmonella typhimurium harbouring TGEV S gene DNA vaccine To investigate the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of TGEV DNA vaccine,the eukaryotic expression plasmid pVAX-S was transformed by electroporation into attenuated Salmonella typhimurium SL7207 and the recombinant was screened and designated as SL7207(pVAX-S).Mouse peritoneal macrophages were infected with SL7207(pVAX-S),the expression of S gene was detected by indirect immunofluorscence.After oral immunization with SL7207(pVAX-S),total cellular RNA was isolated from the terminal ileum of inoculated mice and the transcription of S gene was detected using RT-PCR.mice were inoculated orally with SL7207(pVAX-S) at dosages of 5×10~8 CFU,1×10~9 CFU and 2×10~9 CFU for safty analysis,the results showed that recombinant bacterium was safe to mice at dosage of 2×10~9 CFU.In a vaccination test, mice were immunized orally with recombinant bacterial at dosage of 1×10~7 CFU,1×10~8 CFU and 1×10~9 CFU with three times at two weeks interval,and specific serum IgG and intestinal mucosal IgA antibody were detected by indirect ELISA.The results indicated that mice immunized with different dosages of SL7207(pVAX-S) elicited specific local mucosal and humoral responses as measured by ELISA.The immunogenicity of the DNA vaccine was highly dependent on the dosage of the attenuated bacteria used for oral administration:the dosage of 10~9 CFU group only induced a negligible antibody response at six weeks after immunization,while the dosage of 10~9 CFU group showed highest specific serum IgG and intestinal mucosal IgA antibody response during the experiment and higher(P＜0.05) than 10~8 CFU at week-six after immunization.These results provided preliminary evidence that attenuated Salmonella typhimurium could be utilized as the oral delivery vector for TGEV DNA vaccines.